1. Field of the Invention
The present invention relates to proteins, such as labeled proteins, sensors, and the characterization of analytes in a sample using the proteins.
2. Background of the Related Art
Analyte-binding proteins have many applications. The process of analyte binding often results in useful changes in the protein, such as a change in conformation of the protein.
For example, analyte-binding proteins may be used as sensor proteins to characterize samples. Sample characterization is a broad field that includes, e.g., determining the presence or concentration of analytes, biomedical diagnostics, bioprocessing, and drug screening.
Analyte measurement/detection has many applications. For instance, the increasing demand for the production of important biological products by eukaryotic cell cultures has intensified efforts in the development of sensing devices for monitoring nutrient levels, available oxygen, and cell density in bioreactors. For example, glutamine is a major source of nitrogen and carbon in cell culture media. Glutamine sensing is important in small and large-scale bioprocesses involving eukaryotic cell culture.
Glutamine is considered together with glucose as a limiting factor in cell growth and product yield. Additionally, unfavorable levels of glutamine can lead to the deleterious production of ammonia, which is toxic to cell cultures. Monitoring of glutamine concentrations is therefore an important aspect of process control.
Currently available glutamine biosensors tend to rely on enzymes such as glutaminase (EC 3.5.1.2) in combination with glutamate oxidase (EC 1.4.3.11). Glutamate oxidase is required to suppress the interference from glutamic acid. In another assay, glutamine reacts with three different enzymes to produce NADH, which is then determined spectrophotometrically. High-pressure liquid chromatography and LCMS-MS have been used, but these techniques both require expensive instrumentation. Near-infrared (NIR) spectroscopy allows for noninvasive quantification of glutamine but requires the generation of an elaborate calibration model.
Binding proteins have also been used to measure sugars and sugar derivatives, such as glucose. Glucose is the major carbon and energy source in cellular metabolism. The lack of glucose in a medium will severely limit cell growth and product yield in industrial bioprocess applications. But excessive glucose can also be detrimental, leading to lactate formation via the glycolytic pathway. Therefore, glucose monitoring and control is important for healthy growth of cells and maximum product formation in bioprocesses.
Another example of analyte measurement involves glucose measurement for diabetes treatment. To control the long-term complications associated with diabetes, blood glucose levels must be tightly regulated. This requires careful monitoring of blood glucose.
In view of the above, binding proteins have been utilized as sensors for various analytes including glucose, maltose, phosphate, and glutamine. One advantage of the binding proteins as sensors is that unlike enzymes, they do not require additional reagents. The key event that accompanies molecular recognition between a binding protein and its substrate is a conformational change.
Most binding proteins have a two-domain structure connected by a hinge, as disclosed in QUIOCHO, Phil. Trans. Roy. Soc. London, ser B, 326:341-351 (1990), which is incorporated by reference herein. The binding site is in the interface of the two domains. It has been shown in a number of proteins that binding of the ligand can induce a large conformational change, from an “open” ligand-free structure to a “closed” ligand-bound structure, as disclosed in HSIAO et al., J. Mol. Biol., 262:225-242 (1996); and SUN et al., J. Mol. Biol., 278:219-229 (1998), both of which are incorporated by reference herein. By introducing single fluorophores into such proteins, this conformational change upon ligand binding can be taken advantage of to construct biosensors that respond to their respective ligands. These binding proteins include glucose-binding protein (GBP) MARVIN et al., J. Am. Chem. Soc., 120:7-11 (1998); SALINS et al., Anal. Biochem., 294:19-26 (2001); and TOLOSA et al., Anal. Biochem., 267:114-120 (1999), which are all incorporated by reference herein), maltose-binding protein (GILARDI et al., Anal. Chem., 66:3840-3847 (1994); and GILARDI et al., Prot. Eng., 10(5):479-486 (1997), both of which are incorporated by reference herein), phosphate-binding protein (BRUNE et al., Biochemistry, 33:8262-8271 (1994), which is incorporated by reference herein), and glutamine-binding protein (GlnBP) (DATTELBAUM et al., Anal. Biochem., 291:89-95 (2001), which is incorporated by reference herein).
The choice of labeling sites is generally based on the identification of specific sites on the protein that undergo maximum conformational change upon substrate binding, as disclosed in MARVIN et al., J. Am. Chem. Soc., 120:7-11 (1998), which is incorporated by reference herein. However, this approach neglects the effects on the dye conjugated to the protein. Thus, it is common to observe different environment-sensitive dyes conjugated to the same site showing varying response to analyte concentrations, as disclosed in DATTELBAUM et al., Anal. Biochem., 291:89-95 (2001); and MARVIN et al., J. Am. Chem. Soc., 120:7-11 (1998), both of which are incorporated by reference herein. Consequently, there is a degree of empiricism in the design of binding protein-based biosensors.
One approach to biosensors involves genetically engineering a protein for site-specific positioning of allosteric signal transducing molecules. Structural principles are used to take advantage of cooperative interactions between the signaling molecule and analyte binding. This technique has been applied to maltose binding protein and glucose/galactose binding protein of Escherichia coli (GGBP), as disclosed in MARVIN et al., Proc. Natl. Acad. Sci. USA, 94:4366-4371 (1997); and MARVIN et al., J. Am. Chem. Soc., 120:7-11 (1998), both of which are incorporated by reference herein.
Structural studies of GGBP reveal two domains, the relative positions of which change upon the binding of glucose, as disclosed in CAREAGA et al., Biochem., 34:3048-3055 (1995), which is incorporated by reference herein. Such conformational changes result in spectral changes of environmentally sensitive probes, or changes in the transfer efficiency between donor and acceptor pairs covalently bound to the protein.
U.S. Pat. No. 6,521,446 to HELLINGA, which is incorporated by reference herein, discloses a glucose biosensor comprising a genetically engineered glucose binding protein that includes environmentally sensitive reporter group(s). This document discloses several reporter groups, including osmium(II) bisbipyridyl complexes.
DATTELBAUM et al., Anal. Biochem., 291:89-95 (2001), which is incorporated by reference herein, discloses that labeling a cysteine in position 179 of a glutamine-binding protein with a polarity-sensitive probe, such as acrylodan, results in changes in the fluorescence properties of the probe in response to glutamine. This document discloses that the phase angle was measured at 110 MHz.
TOLOSA et al., Anal. Biochem., 267:114-120 (1999); and LAKOWICZ et. al., Anal. Chem., 70:5115-5121 (1998), both of which are incorporated by reference herein, describe a method in which a labeled glutamine-binding protein is used with an external reference—a long-lived metal-ligand complex—that was added to the solution or applied to the walls of a cuvette. These documents disclose that the combined emission of the labeled protein and the metal-ligand complex allowed for the detection of modulation changes at lower frequencies.
U.S. Pat. No. 6,197,534 to LAKOWICZ et al., which is incorporated by reference herein, discloses engineered proteins for analyte sensing. This document discloses that mutant glucose/galactose binding proteins may have attached fluorophores with widely spaced lifetimes, permitting modulation-based glucose sensing. This document discloses an embodiment in which a long lifetime metal-ligand complex is painted on the outside of a cuvette.
ZHOU et al., Biosens. Bioelectr., 6:445-450 (1991), which is incorporated by reference herein, discloses the immobilization of maltose binding protein (MBP) labeled with IAEDANS. MBP-IAEDANS was immobilized onto PCG by glutaraldehyde coupling, carbodiimide coupling, and diazonium coupling.
WENNER et al., Proc. SPIE, 4252:59-70 (2001), which is incorporated by reference herein, discloses immobilizing phosphate-binding protein (PBP) labeled with N-[2-(1-maleimidyl)ethyl]7-diethylaminocoumarin-3-carboxamide (“MDCC”) in sol-gel.
There remains, however, a need for improved proteins, sensors, and methods for characterizing samples.